Pyromark gold q24 kit

Subsequent bisulphite conversion of 500ng of genomic DNA was carried out according to the manufacturer's instructions [34] [35] (link)[36] (link) using the EZ DNA methylation kit (Zymo Research Corporation, California). PCR was then carried out under the following conditions: initial hot start, 95⁰C for 15 minutes, followed by 45 cycles of 94⁰C for 30 seconds, 56⁰C for 30 seconds, 72⁰C for 30s and a final elongation of 10 minutes at 72⁰C. The PCR products were subsequently electrophoresed on a 1% (w/v) agarose gel electrophoresis to check the size of DNA fragments and also as a quality control measure to check samples for contamination. DNA methylation levels in samples were analysed using the PyroMark Q24 pyrosequencing instrument (Qiagen, UK). Enzymes, substrates and nucleotides from the PyroMark Gold Q24 kit (Qiagen UK) were used. Built in controls within assays to be analysed were used to verify bisulphite conversion. Levels of methylation at each CpG site were analysed using the PyroMark Q24 software [37, 38] (link). The degree of methylation at each CpG site is expressed as the percentage of methylated cytosine over the sum of methylated and unmethylated cytosine.